CN116121259A - A gene MdMYB21 regulating the acid content of apple fruit and its application - Google Patents

Abstract

The invention discloses an apple low-acid regulatory gene MdMYB21, wherein a nucleotide sequence table is shown in a sequence table 1; a recombinant overexpression vector containing apple low-acid regulation gene MdMYB21, application of the apple low-acid regulation gene MdMYB21 in regulation of apple acidity, application of the apple low-acid regulation gene MdMYB21 in obtaining transgenic low-acidity apple material, application of the apple low-acid regulation gene MdMYB21 in obtaining transgenic low-acidity tomato and analysis of influence of 2bp variation in SSR motif (CT) n of promoter of the gene MdMYB21 on malic acid content in Qin Guan x honey crisp hybridization groups; the apple low-acid regulation gene MdMYB21 can negatively regulate the accumulation of malic acid in apple.

Description

A gene MdMYB21 regulating apple fruit acid content and its application

technical field

The invention belongs to the technical field of biological gene engineering, and relates to a gene MdMYB21 for regulating the acid content of apple fruit and its application.

Background technique

Apple (Malus domestica Borkh.), as one of the fruits with the largest planting area and the highest consumption in the world, is rich in minerals and vitamins and has high nutritional value. Improving fruit quality is one of the most important goals in apple breeding programs. As an important part of fruit flavor, the types and contents of soluble sugars and organic acids play a vital role in the formation of fruit sensory quality. The acidity of fruit cells is mainly due to the accumulation of organic acids in the vacuoles, malic acid and citric acid are the main organic acids in most ripe fruits, and malic acid is the main organic acid in apples. Therefore, understanding the mechanism of malic acid accumulation in apple fruit cells is an important aspect to improve apple fruit quality.

Currently, another approach to identify candidate genes for quantitative traits in plant genomes is to discover complex traits of interest through association mapping analysis. Association mapping, also known as linkage disequilibrium (LD) mapping, is a method for observing and identifying genetic polymorphisms that are significantly associated with phenotypic variation using existing breeding and cultivar lines. Fruit acidity is a quantitative trait. Studies have shown that there is a locus that significantly affects fruit acidity at the top of the apple 16 linkage group, named Ma locus, and a main gene that controls fruit acidity has been found at this locus. ALMT9(Ma1), but this gene can only explain about 7.46% of the phenotypic variation in malic acid content observed in mature fruits, suggesting that there may be other candidates controlling apple fruit acidity at this locus Gene. Therefore, this study is of great significance for further mining the genes related to apple fruit acidity by using QTL mapping combined with association analysis.

Although some studies have been done on the location of candidate genes related to apple fruit acidity, the accumulation of fruit acidity is a complex mechanism that requires us to continue to dig further. The research and development of key genes and molecular markers related to apple fruit acidity are of great importance for apple breeding. significance.

Contents of the invention

The purpose of the present invention is to provide a gene MdMYB21 related to the low-acid regulation of apple fruit, which can negatively regulate the accumulation of malic acid in apple fruit.

The present invention also provides a MYB transcription factor, which can negatively regulate the accumulation of malic acid in apple fruit;

The present invention is based on the development of the Ma locus gene label at the top of Apple No. 16 linkage group and its application in the population, as shown in Figure 1; a mixed linear model is used to analyze the correlation between molecular markers and malic acid content, and to screen candidates application of genes.

The technical solution adopted in the present invention is that the apple fruit low-acid regulation gene MdMYB21, and the nucleotide sequence list of the MdMYB21 gene is shown in Sequence Table 1.

The second technical solution adopted by the present invention is a recombinant overexpression vector containing the apple low-acid regulation gene MdMYB21.

The invention clones the apple low-acid regulation gene MdMYB21 in apples, uses homologous recombination to construct overexpression and interference vectors, uses transgenic technology, uses apple callus and fruit as materials for genetic transformation, and obtains transgenic low-acidity traits Application of Apple materials; specifically:

The third technical solution adopted by the present invention is an application of the apple low acid regulation gene MdMYB21 in regulating the acidity of apple fruit.

The fourth technical solution adopted in the present invention is an application of obtaining transgenic low-acid apple material by using the apple fruit low-acid regulation gene MdMYB21.

The fifth technical solution adopted by the present invention is that the apple low-acid regulation gene MdMYB21 is cloned from apples, the overexpression vector is constructed by homologous recombination, and it is heterologously transformed into tomato to obtain transgenic low-acid tomato Applications.

The sixth technical solution adopted in the present invention is the effect of 2bp variation in the gene MdMYB21 promoter SSR motif (CT)n in the hybrid population of 'Qinguan' x 'Honeycrisp' on the content of malic acid.

The beneficial effect of the present invention is

The present invention uses QTL mapping combined with association analysis to identify a gene related to apple fruit acidity. The present invention is an apple low-acid regulation gene MdMYB21, which can negatively regulate malic acid, inhibit the accumulation of malic acid in the vacuole, and improve the quality of apple fruit. Quality plays an important role. An apple low-acid regulation gene MdMYB21 of the present invention can carry out assisted selection breeding through markers in advance, and screen the expected progeny in the hybrid progeny group, shortening the breeding and screening period.

Description of drawings

Figure 1 is an interval correlation analysis diagram of the acidity traits at the Ma site of apple No. 16 staining;

Fig. 2 is a schematic diagram of the genotype detection of the WBG82 marker in natural populations and the correlation between genotype and malic acid content;

Figure 3 is a schematic diagram of the correlation analysis between MdMYB21 expression and malic acid content during apple fruit development;

Fig. 4 is a schematic diagram of specific analysis of MdMYB21 of the present invention in different tissues of apple;

Fig. 5 is the subcellular localization diagram of MdMYB21 of the present invention;

Fig. 6 is the effect of MdMYB21 cloned in the present invention overexpressing in apple callus on malic acid content;

Fig. 7 is the effect of overexpression of MdMYB21 cloned in the present invention on malic acid content in apple fruit;

Figure 8 is a schematic diagram of the influence of overexpression of MdMYB21 cloned in the present invention on total acid content in tomato;

Figure 9 is a schematic diagram of the influence of the 2bp variation in the MdMYB21 promoter SSR motif (CT)n cloned in the hybrid population of 'Qinguan' x 'Honeycrisp' on the content of malic acid.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

The nucleotide sequence of apple low-acid regulation gene MdMYB21 is shown in Table 1.

The extraction and mensuration of embodiment 1 organic acid

The extraction and determination of the organic acid content adopts high performance liquid chromatography (HPLC). The specific determination method is as follows. Take about 0.15 g of the sample, dissolve it in 1 mL ddH ₂ O, place it in water for ultrasonication, and ultrasonicate the mixture at room temperature Treat for 30 minutes, then centrifuge at 12,000 rpm for 15 minutes at 4°C; take the supernatant and filter it with a 0.22 μm water filter. After filtration, the solution can be stored at -80°C;

The organic acid content was determined using an Agilent 1260 Infinity HPLC system (Milford, MA, USA) coupled with a diode array; the mobile phase was 0.02M KH ₂ PO ₄ solution (pH=2.4) at a flow rate of 0.8 mL/min. The column model used is C18-WP, 100A, 4.6ⅹ150mm, 5um, and the detection wavelength is 210nm.

Typing detection of embodiment 2 SSR molecular marker

Take 50-100mg of apple leaf tissue for the extraction of plant genomic DNA; use a 25ul reaction system for the molecular marker PCR reaction solution, as follows: SE008 2×T5 Super PCR Mix: 12.5ul, 10uM Primer pair 1ul, DNA template: 50- 100ng, filled with ddH ₂ O to 25ul; reaction program: 98°C for 3min, {98°C for 10s, 60°C for 10s, 72°C for 5s} 35 cycles, 72°C for 5min; PCR product denaturation: take 5ul of PCR product and add Equal volume of Loading buffer, then 98°C for 10 minutes;

Polyacrylamide gel electrophoresis detection: Pipette 1ul of the denatured PCR product on an 8% polyacrylamide gel for electrophoresis detection, the voltage is constant at 80w, the electrophoresis buffer is 1ⅹTBE, and the electrophoresis time is 1-1.5h;

Silver staining: Separate the glass plate after electrophoresis, wash the disassembled gel three times with distilled water, stain in silver nitrate solution (1.3g/L dissolved in distilled water), and stain for 10min until a clearly visible The strips were then rinsed in distilled water and saved for photographing.

Table 1 Primer list for molecular marker genotyping

Example 3

The relationship between the expression pattern of apple low acid regulation gene MdMYB21 and the accumulation of malic acid:

Take 'Honeycrisp' apple fruit samples 60 days, 90 days, and 120 days after flowering for determination of malic acid content, and each sample includes three biological replicates; take 'Honeycrisp' plant roots, shoot tips, mature leaves, Flowers at the full bloom stage, and fruits at the mature stage, each sample includes three biological replicates for qRT-PCR analysis;

As shown in Figure 3 and Figure 4, the expression level of MdMYB21 is the highest in the fruit; its expression level gradually increases with the development of fruit, and has a significant negative correlation with the content of malic acid; the above results show that the accumulation of MdMYB21 and malic acid is significantly relevant.

Example 4 Subcellular localization of apple low-acid regulation gene MdMYB21

The subcellular localization of MdMYB21 uses the pCAMBIA2300-GFP vector. The GFP contained in the vector is at the C-terminus of the target gene. The selected enzyme cutting sites are KpnI and BamHI. The method of homologous recombination is used. The specific steps are as follows:

(1) The primer sequence is as follows:

KpnI-F: acgggggacgagctcggtaccATGGCTGCTCCTACAACCCCAA

BamHI-R:ggtgtcgactctagaggatcc CGGCCCATCCATGTTCC

(2) Amplify the DNA sequence with the above primers and detect on 1% agarose gel. After the size and brightness of the target band meet the requirements, use the gel recovery kit and buy it from Shanghai Huiling Biotechnology Co., Ltd. The specific operation steps are according to the instructions conduct;

(3) The pCAMBIA2300-GFP vector was digested at 30°C and 37°C for 1 hour, then heat-inactivated at 85°C for 15 minutes, and then stored at 4°C; the digested vector and the recovered target gene fragment were used for homologous recombination Enzyme for ligation, enzyme cutting system and ligation system are as follows:

Table 2 Double Enzyme Digestion System

According to the reaction procedure of 30°C for 1h, 37°C for 1h, 85°C for 15min, and 4°C.

Table 3 Enzyme digestion product ligation system

Check whether the target gene is constructed on the carrier by PCR. The size of the MdMYB21 fragment is 894bp. After the band size is consistent, the bacterial solution is sent to the company for sequencing. The sequencing result is compared with the downloaded sequence. If the sequence is consistent, the connection is successful. A recombinant vector containing MdMYB21 was obtained, named MdMYB21-GFP, and pCAMBIA2300-GFP empty vector was used as a control, and the two were respectively introduced into Agrobacterium strain GV3101 and injected into tobacco leaves, and observed under a confocal microscope; as shown in Figure 5 The results show that the green fluorescence of the empty vector can be clearly observed on the cell membrane and nucleus, indicating that the empty vector is good; when the green fluorescence of the target gene is observed, it is clearly expressed in the nucleus, indicating that MdMYB21 is localized in the nucleus.

Example 5 Effect of overexpression of MdMYB21 cloned by the present invention on malic acid content in apple callus, fruit and tomato:

In order to further study the influence of the MdMYB21 gene on the accumulation of malic acid in the present invention, the transgenic callus and apple fruit transferred to the pMDC83 empty vector were used as controls; As shown in Example 1, the detection results are shown in Fig. 6, Fig. 7, and Fig. 8; Fig. 6, 7, and 8 analysis results show that in apple callus, overexpression of MdMYB21 in fruit and tomato can significantly reduce Malic acid content;

Example 6 The influence of the 2bp variation in the MdMYB21 promoter SSR motif (CT)n cloned in the 'Qinguan' x 'Honeycrisp' hybrid population of the present invention on the content of malic acid:

The present invention uses the DNA of 'Qinguan' and 'Honeycrisp' hybrid populations as a template, uses the following primers as the MdMYB21 promoter region to be amplified, and the amplified PCR product is sequenced; the malic acid content is determined according to the examples 1 as shown. The result is shown in Figure 9.

PCR primers are as follows:

MYB21-F: CCATGGACTATACAACAAAAATTTC

MYB21-R:AGCTGCTCTCTTGCTTGAGAGAGA

Sequence Listing 1

Nucleotide sequence

ATGGCTGCTCCTACAACCCCAAATGAAGAAAATGAGTTCAGAAGAGGGCCATGGACTCTTGAGGAAGACAATCTGCTTATACATTACATCGTGAACCACGGCGAAGGCCATTGGAATTCTGTAGCAAAACTTGCAGGATTGAAGAGGACCGGAAAAAAGCTGCAGATTGAGATGGCTAAATTACTTAAAACCCGACATTAAGCGCGGGAACCTTACTCCG CAAGAACAGCTCATGATCCTTGAACTCCACTCCAAGTGGGGTAACAGGTGGTCTAAAATTGCGCAGCATTTGCCGGGAAGAACAGACAATGAGATAAAGAACTATTGGAGAACAAGGGTGCAAAAACAGGCGCGCCAACTGAACATCGAGTCGAACAGCGAACAATTTCGATGCAGTTCGGGGTTTCTGGGTGCCGACTCTGCTGCAAAAAAATGGA GCAATCTTCTTCTTCTTGTTCTTCAACCTTTGAGCACTTCTCAGAACTCCGCATCTCCTTGTCTGTCACCAAATCACGCAGCTCCTTCCGTGCCACTCTCAACCTCTCCACCTAGCAATGCGACAAACGTGTTGGACAATTATCACATTAGTGGAAATTCCAATCTTGCCACCGTCCCAAAGTAATATCCTTTCGGCGGATTCTTTTGTTTCACACGTGCCTCAAATGGCAGAA CCGTCCACGAGTTTTTCCCCCTGCATATTACCGACTTGGCTACAGCAGCTTAAGTCCAGATGGCAGTCACTACGTGGACAGCAGTAGCTATGACGTGGAGGGTCTCAGCCTGGACCCTGTTTCGGCAATGGGCAATCTTGGCAATTCACAGTTTGATTGCCAGATGGGGGGAAATGATTGGATGTTGGACAATGTGACTGACAGTTTATGGAACAT GGATGGGCCGTGA

Claims (6)

1. Apple fruit low-acid regulation gene MdMYB21, characterized in that the nucleotide sequence list of the MdMYB21 gene is shown in Sequence List 1. 2. A recombinant overexpression vector containing the apple low-acid regulation gene MdMYB21 according to claim 1. 3. an application of the apple low-acid regulation gene MdMYB21 according to claim 1 in regulating apple fruit acidity. 4. an application of obtaining transgenic low-acidity apple material with the apple fruit low-acid regulation gene MdMYB21 according to claim 1. 5. An application of obtaining transgenic low-acidity tomato with the apple fruit low-acid regulation gene MdMYB21 according to claim 1. 6. Analysis of the influence of the 2bp mutation in the SSR motif (CT)n of the gene MdMYB21 promoter on the content of malic acid in Qinguan x Honeycrisp hybrid population.

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